Ferroptotic vulnerability in M1 and M2 macrophages is associated with adaptive radiation resistance in esophageal adenocarcinoma

Background: Radiation resistance remains a critical barrier to enhancing cures after chemoradiation therapy (CRT) in esophageal adenocarcinoma (EAC). Ferroptosis, a lipid peroxidation-driven cell death pathway, is increasingly recognized as a regulator of tumor-immune interactions. We investigated how ferroptotic susceptibility of macrophages might differ in radiation responders (GR) and non-responders (NR) in EAC patients using both single cell sequencing and spatial proteomic analysis. Methods: scRNA-seq and sequential multiplex-IF using Lunaphore COMET were performed on patient biopsies before, during and after CRT to characterize cellular subsets and ferroptosis markers at single-cell resolution. In vitro ferroptosis assays were performed in THP-1, and human PBMC-derived M1/M2 macrophages following 0-12 Gy radiation ± RSL3 or ferrostatin. Ferroptotic activity was assessed by CellTiter-Glo, BODIPY-C11 oxidation, and GPX4 staining (Confocal, flowcytometry). Results: scRNA-seq confirmed robust myeloid cell expansion in both GR and NR and revealed that pro-ferroptosis gene programs (BH4 biosynthesis, iron utilization and glycolysis pathway) were upregulated in NR both at baseline and during CRT, particularly in the myeloid population, which greatly expands during CRT. Conversely, anti-ferroptosis pathways (GPX4) were enriched in GR and suppressed in NR. COMET profiling revealed that non-responders (NR) displayed higher baseline M2 macrophage density and significantly elevated ferroptosis marker 4-HNE in both M1 and M2 subsets. During CRT, NRs exhibited a further increase in M2 macrophage infiltration, reinforcing an immunosuppressive TME, whereas good responders (GR) maintained higher M1 representation. In vitro, radiation significantly potentiated RSL3-induced ferroptosis in both human and murine macrophage-like cells; ferrostatin rescued viability, confirming ferroptotic cell death. Human macrophages displayed polarization-dependent susceptibility: M2 macrophages were sensitive to radiation-induced ferroptosis, while M1 macrophages were resistant Conclusion: CRT response appears to relate to macrophage polarization and ferroptosis susceptibility. Non-responders are characterized by (i) M2-dominant macrophage landscapes, (ii) elevated lipid peroxidation, (iii) expansion of ferroptosis-primed myeloid subsets, and (iv) persistent activation of pro-ferroptosis pathways. Collectively this data ascribes macrophage ferroptosis as a potential targetable axis for radio-sensitization in EAC.